European Genome-Phenome Archive

File Quality

File InformationEGAF00000140856

File Data

Base Coverage Distribution

This chart represents the base coverage distribution along the reference file. Y-axis represents the number of times a position in the reference file is covered. The x-axis represents the range of the values for the coverage.

Data is represented in a log scale to minimise the variability. A high peak in the beginning (low coverage) and a curve descending is expected.

28 200 06835 790 52451 797 00275 243 682104 958 677138 394 079171 513 263200 315 358221 073 080231 765 671231 579 175221 552 090203 591 547180 309 825154 421 688128 203 802103 508 26881 360 96362 540 15047 120 87234 949 26725 588 01418 583 83513 477 4659 793 9327 171 4625 318 9324 003 9143 075 0892 395 1561 886 5521 503 2871 207 555983 569801 995662 025543 086450 402377 366319 365273 156236 643206 486185 339166 535151 451139 458129 334120 636113 061107 166100 45694 78389 42284 22179 66774 35870 30965 76061 17057 59954 93751 48048 86246 03642 97240 84138 19336 02834 65932 66031 26729 77828 57326 90225 74424 38923 38422 29621 14720 58819 79019 12218 21818 17017 29516 58615 99815 38714 53014 05913 74313 32612 80612 70412 47511 95811 62311 19510 99510 34310 0989 9889 4049 4969 2909 0348 6548 4048 3078 1768 1557 7657 5137 3037 0236 7326 5316 3156 2996 0905 7515 5095 4645 3344 8284 8834 7854 6694 5694 3474 0973 9833 9503 9313 6973 6793 5943 5433 5443 3283 2103 1493 1093 0422 9632 9822 8792 7822 6842 7252 6702 5302 5882 5082 4362 4122 4592 4842 3862 3732 4192 4052 4082 2732 2732 2522 2012 1552 0832 0582 0222 0041 9321 8711 8071 8911 7751 9291 9341 8021 8021 7671 6581 7421 6801 6901 6631 5671 6291 5741 6701 5451 5881 5611 5291 4441 3591 4461 4051 4571 4271 4691 4141 4841 4491 4021 3511 3251 3191 3051 2161 2631 2521 2571 2291 2421 2421 1361 2271 1291 1471 2091 1771 1811 1241 1041 1911 2191 1301 0981 1171 1741 1661 2011 2271 0621 0971 1931 0901 1761 0691 0961 0851 0421 0331 0341 0751 0451 0101 1019489579049919519859779199449278788928769428998988458288469478477518287927668038377838338358787437717937787267847467187337767027377407337757197587146687347087486947467647747007447566957266797387036956657426616826866906996836856596316576236456236456646786026456176196206616065675906175945785636005485695485925856215445655755735535024924995185145045145165035295075285055025155495075264965295174974914774275195284624304534344184404434514184464574244314104544254433984144024314143814484234314153974194344384524294193994273893703793403673553893973954133743773983563663713534033873423793823543633533253703123523773103163473823193313273383313343213133173293133153143062982843032662822993113073192793013122873253042973483393572792823132492892712602802682712912682823112862902692603072882762803093092872972822872612892232492802372402462672352802472892632852672922542602812412382692672582532572712442342822322493002782942432452682252402312312672202382272272142622442502362462182352322252221942332012112482022662032142432422322662282032502042341901891821812021752052141922062102322272122161902121962111932121982042052042171881961932102101841881841911771781881871581701862051991871862191681571871951881831832102201971771911911861781841851791931812232221952081701861781911891821671781751921801821621791741471781841581661741701691661761581711721831881511641731531731581561761581791451731631591711681531551601781661551561311571801661491251511311451601431521621561641471431551501481491191411311421401381501341561351351231311331541311291441311401281431421331541411401571251401501641341351661451301401371281461751321011141351111271101451231651331431291301581291281251231371201321281381281311301261351611501301611381261371221241261381181351281171561371371301351341241231251601341331111281091201501361301411231191161161091001241321121101121291231021061069011292981021069510110994101861041181001068610788881179591981221061111069110110710192971051008910110112111212311412311812610010485108969312295117112115109113115123105104979587117981091221051148810892919794841099292927610387104100828810110991978768891141001067885100106999811093878610395869495110107771071231019711410494105851048894978092911021069185121 504100200300400500600700800900>1000Coverage value1001k10k100k1M10M100M# Bases

Base Quality

The base quality distribution shows the Phred quality scores describing the probability that a nucleotide has been incorrectly assigned; e.g. an error in the sequencing. Specifically, Q=-log10(P), where Q is the Phred score and P is the probability the nucleotide is wrong. The larger the score, the more confident we are in the base call. Depending on the sequencing technology, we can expect to see different distributions, but we expect to see a distribution skewed towards larger (more confident) scores; typically around 40.

13 507 132891 4162 284 4697 597 47613 638 2076 051 36876 709 62491 085 12222 489 48317 751 25817 516 662136 406 268172 330 56897 324 57645 753 16939 161 10852 765 81623 102 95225 492 77517 661 26414 087 92532 253 942124 461 943222 239 778477 756 350254 839 373186 480 614182 857 290190 595 169154 834 739269 570 258573 497 123609 258 919867 526 7641 199 701 0061 883 331 4804 671 687 6119 039 932 6559 408 374 1632 925 356 554431 986 53388 553 38710 568 5874 312 32400510152025303540Phred quality score0G1G2G3G4G5G6G7G8G9G# Bases

Mapped Reads

Number of reads successfully mapped (singletons & both mates) to the reference genome in the sample. Genetic variation, in particular structural variants, ensure that every sequenced sample is genetically different from the reference genome it was aligned to. Small differences against the reference are accepted, but, for more significant variation, the read can fail to be placed. Therefore, it is not expected that the mapped reads rate will hit 100%, but it is supposed to be high (usually >90%). Calculations are made taking into account the proportion of mapped reads against the total number of reads (mapped/mapped+unmapped).

96 %333 070 82996 %4 %

Both Mates Mapped

When working with paired-end sequencing, each DNA fragment is sequenced from both ends, creating two mates for each pair. This chart shows the fraction of reads in pairs where both of the mates successfully map to the reference genome. .

Notice that reads not mapped to the expected distance are also included as occurs with the proper pairs chart.

95.3 %330 682 41095.3 %4.7 %

Singletons

When working with paired-end sequencing, each DNA fragment is sequenced from both ends, creating two mates for each pair. If one mate in the pair successfully maps to the reference genome, but the other is unmapped, the mapped mate is a singleton. One way in which a singleton could occur would be if the sample has a large insertion compared with the reference genome; one mate can fall in sequence flanking the insertion and will be mapped, but the other falls in the inserted sequence and so cannot map to the reference genome. There are unlikely to many such structural variants in the sample, or sequencing errors that would cause a read not to be able to map. Consequently, the singleton rate is expected to be very low (<1%).

0.7 %2 388 4190.7 %99.3 %

Forward Strand

Fraction of reads mapped to the forward DNA strand. The general expectation is that the DNA library preparation step will generate DNA from the forward and reverse strands in equal amounts so after mapping the reads to the reference genome, approximately 50% of them will consequently map to the forward strand. Deviations from the 50%, may be due to problems with the library preparation step.

50 %173 517 92650 %50 %

Proper Pairs

A fragment consisting of two mates is called a proper pair if both mates map to the reference genome at the expected distance according to the reference genome. In particular, if the DNA library consists of fragments ~500 base pairs in length, and 100 base pair reads are sequenced from either end, the expectation would be that the two reads map to the reference genome separated by ~300 base pairs. If the sequenced sample contains large structural variants, e.g. a large insertion, where we expect the reads mapping with a large separation would be a signal for this variant, and the reads would not be considered as proper pairs. Based on the sequencing technology, there is also an expectation of the orientation of each read in the fragment.

The rate of proper pairs is expected to be well over 90%; even if the mapping rate itself is low as a result of bacterial contamination, for example.

94.9 %329 245 24894.9 %5.1 %

Duplicates

PCR duplicates are two (or more) reads that originate from the same DNA fragment. When sequencing data is analyzed, it is assumed that each observation (i.e. each read) is independent; an assumption that fails in the presence of duplicate reads. Typically, algorithms look for reads that map to the same genomic coordinate, and whose mates also map to identical genomic coordinates. It is important to note that as the sequencing depth increases, more reads are sampled from the DNA library, and consequently it is increasingly likely that duplicate reads will be sampled. As a result, the true duplicate rate is not independent of the depth, and they should both be considered when looking at the duplicate rate. Additionally, as the sequencing depth in increases, it is also increasingly likely that reads will map to the same location and be marked as duplicates, even when they are not. As such, as the sequencing depth approaches and surpasses the read length, the duplicate rate starts to become less indicative of problems.

12.3 %42 558 01012.3 %87.7 %

Mapping Quality Distribution

The mapping quality distribution shows the Phred quality scores describing the probability that a read does not map to the location that it has been assigned to (specifically, Q=-log10(P), where Q is the Phred score and P is the probability the read is in the wrong location). So the larger the score, the higher the quality of the mapping. Some scores have a specific meaning, e.g. a score of 0 means that the read could map equally to multiple places in the reference genome. The majority of reads should be well mapped, and so we expect to see this distribution heavily skewed to a significant value (typically around 60). It is not unusual to see some scores around zero. Reads originating from repetitive elements in the genome will plausibly map to multiple locations.

23 633 38188 21245 197147 51243 85771 12855 74478 75064 955508 194236 360191 129445 716112 222113 251997 136129 443947 070283 32688 189463 58325 427269 9971 206 25616 766204 52815 72417 14116 67615 922 30422 14517 12218 51423 67419 34826 102737 2564 520 22556 38432 35486 39074 87435 782142 95850 16464 524436 00888 89298 556104 316172 54672 200202 084152 434220 428345 026699 534292 076 838051015202530354045505560Phred quality score20M40M60M80M100M120M140M160M180M200M220M240M260M280M# Reads

Mapped vs Unmapped

Stacked column chart for both mapped and unmapped reads along all chromosomes in the reference file. It is a similar representation as shown in the Mapped reads chart but for each chromosome. Although sequenced sample may be a female, it is possible to get reads in the Y chromosome as there are common regions in both chromosomes called pseudoautosomal regions (PAR1, PAR2).

Unmapped reads belonging to each chromosome are determined when the one mate/pair is aligned and the other is not. The unmapped read should have chromosome and POS identical to its mate. It could also be due when aligning is performed with bwa as it concatenates all the reference sequences together, so if a read hangs off of one reference onto another, it will be given the right chromosome and position, but it also be classified as unmapped.

100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped